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Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network

Nature Cell Biology volume 18, pages 181–190 (2016)Cite this article

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Abstract

Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. Here we describe the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. We identify the Formin family protein Diaphanous (Dia) as the main actin nucleator involved in generating this structure, and uncover Rho as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.

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Figure 1: Dynamics of actin coat formation around vesicles secreting glue proteins.
Figure 2: Dia is recruited to fused vesicles to generate actin coats.
Figure 3: Myosin II is recruited to actin-coated vesicles to facilitate vesicle compression.
Figure 4: Rho1–Rok signalling mediates vesicle compression.
Figure 5: Myosin II forms a ‘cage’-like structure on actin-coated vesicles.
Figure 6: Arp2 knockdown compromises myosin organization.
Figure 7: Sequence of orchestrated events in glue-vesicle secretion.

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Acknowledgements

We thank J. M. Philippe and T. Lecuit for the active Rho1 reporter strain, and the Bloomington and VDRC stock centres for Drosophila lines. We thank S. Levin-Zaidman (Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging, Weizmann Institute of Science) for conducting the electron microscopy studies. We thank E. Bouchbinder and A. Livne for discussions and suggestions on biomechanics and quantification of vesicle secretion. We are grateful to members of the B.-Z.S. laboratory, especially E. Geron, for stimulating discussions. This work was supported by a USA-Israel BSF grant to E.D.S. and B.-Z.S. B.-Z.S. is an incumbent of the Hilda and Cecil Lewis chair in Molecular Genetics.

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Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

    Tal Rousso, Eyal D. Schejter & Ben-Zion Shilo

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  1. Tal Rousso
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  2. Eyal D. Schejter
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  3. Ben-Zion Shilo
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Contributions

T.R. designed the experiments, carried them out and participated in writing the manuscript, E.D.S. and B.-Z.S. designed the experiments and participated in writing.

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Correspondence to Eyal D. Schejter or Ben-Zion Shilo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fusion and membrane mixing in glue-vesicle secretion.

(a–a ′) Time series of a single vesicle secretion event from a glue-secreting salivary gland expressing UAS-LifeAct-GFP under the fkh-Gal4 driver (green, a ′), and injected with Dextran-TMR (red, a). At t = 0, Dextran is restricted to the lumen by the apical membrane, outlined by actin. Dextran is observed to fill the vesicle prior to appearance of an actin coat, demonstrating that fusion precedes coating. (b–b ′) Time series of a single vesicle from a glue-secreting salivary gland expressing both the PI(4,5)P2 sensor UAS-PH-PLCδ-GFP (green, b), and UAS-LifeAct-Ruby (red, b ′) under the fkh-Gal4 driver. In addition to a strong apical membrane localization, PI(4,5)P2 begins to outline the vesicle at 0.32 m, ahead of actin coating, first observed at 0.97 m. (c–c ′) Time series of a single vesicle, from glue-secreting salivary glands expressing the PI(4,5)P2 sensor PH-PLCδ-GFP under the fkh-Gal4 driver (c ′), and injected with Dextran-TMR (c). Dextran is restricted to the lumen by the apical membrane, outlined by PI(4,5)P2. PI(4,5)P2 appears upon the vesicle membrane simultaneously with the appearance of Dextran (at 0.14 m). Scale bars: 5 μm.

Supplementary Figure 2 The number of myosin stripes is weakly correlated with vesicle size.

A graph describing the number of myosin stripes counted for each vesicle, relative to vesicle size (diameter). As demonstrated by the correlation coefficient (0.41, P-value = 0.007, Microsoft Excel), the two are only weakly correlated. n = 38 vesicles.

Supplementary information

Supplementary Information

Supplementary Information (PDF 396 kb)

Glue secretion from ecdysone treated larval salivary glands.

Time-lapse movie of cultured salivary glands expressing UAS-LifeAct-Ruby (red) under the fkh-Gal4 driver, and Sgs3-GFP under endogenous regulatory sequences (green). Movie begins 2 h after the addition of ecdysone to the culture media. GFP is observed within glue granules spanning the entire volume of the cell, and actin is observed enriched at the apical surface. An increase in GFP fluorescence in the lumen is readily apparent over time, concomitant with actin coat structures around apical vesicles, and expansion of the lumen. Movie imaged using 20× lens Plan-Apochromat NA 0.8 with 1.5 digital zoom. Stacks of 6 z planes at 4 μm intervals were taken, with no time interval. Movie was processed post imaging, and the time elapsed is shown within the movie. Scale bar: 20 μm. (MOV 1949 kb)

Hydrostatic pressure in glue vesicles following fusion.

Time-lapse movie of cultured salivary glands expressing UAS-LifeAct-Ruby (red) under the fkh-Gal4 driver, and Sgs3-GFP under endogenous regulatory sequences (green), approximately 1.5 h following secretion onset (3.5 h following addition of ecdysone). The vesicle in the center increases in size and GFP intensity just before it coats with actin and squeezes, indicative of glue that was compressed within the vesicle upon fusion. The lumen is at a deeper focal plane, and the direction of secretion is perpendicular to this optical plane. Movie imaged using 63× lens Plan-Apochromat NA 1.4 with 4 digital zoom, with no time interval. Movie was processed post imaging and the time elapsed is shown within the movie. Scale bar: 2 μm. (AVI 146 kb)

dia mutants exhibit abnormal secretion with no actin coat formation.

Time-lapse movies shown side by side of wildtype (left) and dia5/Df (right) salivary glands expressing the microfilament binding construct Utrophin-GFP under the fkh-Gal4driver. Movie begins 2 h after the addition of ecdysone to the culture media. Wildtype glands exhibit a dynamic appearance of actin coats as the lumen expands, in contrast to dia mutant salivary glands which show no actin coats, and only limited expansion of the lumen. Movies were taken simultaneously using 20× lens Plan-Apochromat NA 0.8 with 1 digital zoom. Stacks of 6 z planes at 4 μm intervals were taken, with no time interval. Movies were processed post imaging and the time elapsed is shown within the movie. Scale bar: 20 μm. (AVI 3654 kb)

zip-RNAi expressing salivary glands exhibit actin coated vesicles that do not contract.

Time-lapse movie of salivary glands expressing zip-RNAi and UAS-LifeAct-Ruby (red) under the fkh-Gal4 driver, and Sgs3-GFP under endogenous regulatory sequences (green). The population of 62 vesicles in this video was analyzed: actin coats are clearly shown forming around glue vesicles, however very few vesicles contract, as monitored by size reduction. The majority of vesicles (60%) lose their actin coat after a few minutes while glue remains within the vesicle and no size reduction is observed (red arrow). Some vesicles (15%) exhibit oscillations in actin assembly with no apparent size reduction or glue secretion (white arrows). Only a fraction of the vesicles exhibit size reduction, however for some of these vesicles fusion and content release to another vesicle take place, rather than to the lumen (purple arrow). Movie imaged using 20× lens Plan-Apochromat NA 0.8 with 1.1 digital zoom. Stacks of 7 z planes at 4 μm intervals were taken, with no time interval. Movie was processed post imaging and shows a single focal plane. The time is shown within the movie. Scale bar: 10 μm. (AVI 4053 kb)

Rok-RNAi expressing salivary glands exhibit actin coated vesicles that do not contract.

Time-lapse movie of salivary glands expressing Rok-RNAi and UAS-LifeAct-GFP under the fkh-Gal4 driver. The population of 58 vesicles in this video was analyzed: actin coats are clearly shown forming around glue vesicles, however very few vesicles contract, as monitored by size reduction. The majority of vesicles (64%) lose their actin coat after a few minutes while glue remains within the vesicle and no size reduction is observed (red arrow). Some vesicles (31%) exhibit oscillations in actin assembly with no apparent size reduction or glue secretion (white arrows). Only a small fraction of the vesicles exhibit size reduction, however for some of these vesicles fusion and content release to another vesicle take place, rather than to the lumen (purple arrow). Movie imaged using 20× lens Plan-Apochromat NA 0.5 with 1.2 digital zoom. Stacks of 10 z planes at 4 μm intervals were taken, with no time interval. Movie was processed post imaging and shows a single focal plane. The time is shown within the movie. Scale bar: 10 μm. (AVI 8612 kb)

Rho-RNAi expressing salivary glands exhibit no actin coats and formation of giant vesicles.

Time-lapse movie of glue-secreting salivary glands expressing Rho1-RNAi and the microfilament binding construct UAS-Utrophin-GFP under the fkh-Gal4 driver, and Sgs3-GFP under endogenous regulatory sequences. The movie begins 2 h after the addition of ecdysone to the culture media. Actin coats are not observed around glue vesicles. Weak actin coats are observed around giant vesicles and a few normal-sized vesicles, which are not secreted. The formation and growth of giant vesicles is readily observed over time. These glands exhibit a widened lumen, due to an early effect on gland cell morphology15,34, unrelated to secretion. Movie imaged using 20× lens Plan-Apochromat NA 0.8 with 1.5 digital zoom. Stacks of 6 z planes at 4 μm intervals were taken, with no time interval. Movie was processed post imaging and the time is shown within the movie. Scale bar: 20 μm. (MOV 416 kb)

Giant vesicles in Rho1-RNAi expressing glands are the result of fusion between multiple glue vesicles.

Time-lapse movie of salivary glands expressing Rho1-RNAi under the fkh-Gal4 driver, and Sgs3-GFP under endogenous regulatory sequences, at 60 m following secretion onset (3 h following addition of ecdysone). Actin coated vesicles are clearly seen fusing with the giant glue vesicle. The lumen is in a deeper focal plane, and the direction of secretion is perpendicular to this optical plane. Movie imaged using 63× lens Plan-Apochromat NA 1.4 with 1.5 digital zoom, with no time interval. Movie was processed post imaging, and the time elapsed is shown within the movie. Scale bar: 5 μm. (AVI 462 kb)

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Rousso, T., Schejter, E. & Shilo, BZ. Orchestrated content release from Drosophila glue-protein vesicles by a contractile actomyosin network. Nat Cell Biol 18, 181–190 (2016). https://doi.org/10.1038/ncb3288

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